Previous work has demonstrated that forkhead transcription factors, which include the FoxO subfamily, play a critical role in muscle atrophy by inducing expression of the atrophy-related ubiquitin ligases. The proliferation of muscle precursor cells (MPC) is also essential for skeletal muscle mass. The hypothesis was tested that the FoxO forkhead transcription factor FoxO3a hinders MPC proliferation. The present studies were designed to determine the effects of overexpression of FoxO3a on in vitro proliferation of MPCs. MPCs infected with an adenovirus for wild-type FoxO3a had decreased DNA synthesis as detected by the incorporation of 5-bromo-2' deoxyuridine. In general, cyclin-dependent kinase inhibitors, including p27(Kip1)and p21(Waf/Cip1), inhibit cell proliferation. Associated with the impaired MPC proliferation, we found an increase in the promoter activity and protein levels of the cyclin-dependent kinase inhibitor p27(Kip1), whereas there was no effect and a decrease in the promoter activity and protein levels of p21(Waf/Cip1). FoxO3a overexpression had no effect on either the phosphorylation of retinoblastoma protein (ser780) or cyclin D1 protein levels, suggesting that FoxO3a does not effect the early phase of the G(1)-S transition. In addition to its ability to induce muscle atrophy, these studies identify FoxO3a as a negative regulator of MPC proliferation. Our findings suggest that attenuating increased FoxO3a may restore MPC proliferation to prevent atrophy and improve the regenerative capacity of skeletal muscle.